**2.2. Virus-like particles as a VADS**

Virus-like particles (VLPs) consist mainly of viral proteins devoid of viral genomes to mimic the natural structure of virions and have been engineered to carry agents for various applications, including, particularly, for constructing a VADS for delivering subunit vaccines based on their viral envelop structures suitable for presenting functional spikes on NP surfaces to maintain the intrinsic immunogenicity apt to trigger immunoresponses [16]. VLPs are usually manufactured using protein expression systems based on bioengineered bacteria, yeast, insect, avian, mammalian or plant cells, or using cell-free protein synthesis system (CFPS), which provide an alternative to construct effective VADSs with beneficial characteristics, such as having defined structure formed through self-assembling, large cargo loading capacity, easy functionalization with ligands, and high stability and low toxicity [17]. As a VADS for producing vaccine candidates, VLPs are often designed with characteristics identical to native virus especially in the aspects of the immunochemical properties, 3-D (3-dimensional) architectures and morphological conformations, through engineering on their particulate structures, which, like the native virus, include the nonenveloped and enveloped types. The nonenveloped VLPs mainly consist of one or more pathogenic components, but do not contain any components of the expression hosts, while the enveloped VLPs generally consist of matrix proteins which are enveloped in a lipid membrane derived from the expression hosts, possibly, with glycoproteins embedded into the bilayered membranes.

VLPs are widely used as a VADS because they possess several clear advantages, including induction of immunity with a broad cross-protection rendering onetime immunization to protect against different virus genotypes, high potency to trigger immune responses providing an option to conquering intractable pathogens such as HIV and HCV (hepatitis C virus), high thermostability possibly requiring no integral cold chain to keep viability favoring global vaccination, and high manufacturing efficiency in large scale while at low cost offering a strategy to handle the emergency arising from infectious diseases, such as Ebola outbreak and epidemic. For example, HCV infection is still a significant public health problem, though it has been partially addressed with the advent of directly acting antiviral agents (DAAs), which represent a major advance toward controlling HCV but confer little protection against reinfection [18]. Presently, around 71 million people in the world are living with chronic HCV infection, and each year nearly half a million of them will die of HCV infection or its complications, rendering it urgent to develop an effective vaccine capable of eradicating HCV, which may well be produced using the VLP-based VADS, in reference to HBV vaccines. Recently, a quadrivalent genotype 1a/1b/2a/3a HCV VLP vaccine was successfully engineered by researchers using scale-up production methods of Huh7 cell factories containing a recombinant adenoviral expression system representing each HCV genotype, followed by cell lysing and purification with iodixanol ultracentrifugation and stirred cell ultrafiltration [19]. When given subcutaneously to mice, whether in the presence of an adjuvant (system) or not, the quadrivalent vaccine consistently induced production of Ab and nAb (neutralizing Ab) together with robust T and B cell responses for eliciting broad humoral and cellular immunity, indicating the VLP-based VADS a useful tool which may be employed for the production of an effective HCV vaccine [20].

that among different particles with a size ranging from 20 to 2000 nm, 40 nm NPs induced the strongest cellular and humoral immunity [12]. Further investigations demonstrated that covalent linkage of peptide to NPs is a requirement for eliciting immunization efficacy and also proved that 40-nm-sized NPs serve as a VADS owing to their preferential uptake by APCs and their ability to traffic to lymph nodes to induce strong immune responses compared to their

Notably, Schöttler et al*.* recently reported the counterintuitive research results on cellular uptake of NPs with PEGylation (modification with polyethylene glycol), which is gold standard in removal of immune clearance of in vivo NPs through the mechanism of reducing nonspecific cellular uptake of nanocarriers [14]. The researchers documented that polystyrene NPs, which had been modified with PEG or poly(ethyl ethylene phosphate) (PEEP), only had been exposed to plasma proteins, could exhibited a lowered cellular uptake by macrophages (RAW264.7 cells), whereas those not exposed to plasma proteins showed high nonspecific uptake. Further mass spectrometric analysis revealed that the plasma-exposed nanocarriers formed a protein corona which was identified to contain just an abundance of clusterin proteins (known as apolipoprotein J) and to be the decisive factor controlling lowered nonspecific cellular uptake of the PEGylated or PEEPylated polystyrene NPs, and to contrast, the classic conception that PEGylated NPs free of immune clearance is resulted from avoidance of protein adsorption. These outcomes indicated that PEG as well as PEEP can affect the composition of protein adsorption by polystyrene NPs, and that the presence of certain type proteins may be just a prerequisite in preventing nonspecific cellular uptake of NPs, defying the conventional belief that PEGylation reduces protein adsorption thereby conferring a stealth effect [15].

Virus-like particles (VLPs) consist mainly of viral proteins devoid of viral genomes to mimic the natural structure of virions and have been engineered to carry agents for various applications, including, particularly, for constructing a VADS for delivering subunit vaccines based on their viral envelop structures suitable for presenting functional spikes on NP surfaces to maintain the intrinsic immunogenicity apt to trigger immunoresponses [16]. VLPs are usually manufactured using protein expression systems based on bioengineered bacteria, yeast, insect, avian, mammalian or plant cells, or using cell-free protein synthesis system (CFPS), which provide an alternative to construct effective VADSs with beneficial characteristics, such as having defined structure formed through self-assembling, large cargo loading capacity, easy functionalization with ligands, and high stability and low toxicity [17]. As a VADS for producing vaccine candidates, VLPs are often designed with characteristics identical to native virus especially in the aspects of the immunochemical properties, 3-D (3-dimensional) architectures and morphological conformations, through engineering on their particulate structures, which, like the native virus, include the nonenveloped and enveloped types. The nonenveloped VLPs mainly consist of one or more pathogenic components, but do not contain any components of the expression hosts, while the enveloped VLPs generally consist of matrix proteins which are enveloped in a lipid membrane derived from the expression hosts, possibly, with glycoproteins embedded into the bilayered membranes. VLPs are widely used as a VADS because they possess several clear advantages, including induction of immunity with a broad cross-protection rendering onetime immunization to

larger counterparts [4, 13].

30 Immunization - Vaccine Adjuvant Delivery System and Strategies

**2.2. Virus-like particles as a VADS**

Globally, HIV continues to be a major public health issue and also claims approximately 1 million lives each year, although total people living with HIV have the opportunity to receive antiretroviral drugs (ARVs), which may effectively control the virus from transmission and causing illness but are also found to be undermined in efficacy by pathogens that had evolved with drug resistance [21]. Still, a highly effective vaccine is believed to be the ultimate weapon able to erase HIV and associated disease AIDS (acquired immune deficiency syndrome), though at present, there are no such a product in markets and, moreover, many products developed in previous years bearing such expectations failed to show clinical efficacy in fighting this rapidly mutating pathogen, including especially the big trial, known as STEP, which was halted in 2007 after the vaccine was found to increase the risk, instead of prophylaxis, of HIV infection [22]. Nevertheless, scientists are getting closer than ever to developing such an effective product, as evidenced by a large-scale clinical study conducted in 2009 in Thailand (called RV144) showing that immunization with a combination of two HIV vaccines prevented about 31% of new infections through a prime-boost combination regime [23], which comprises four priming intramuscular injections of ALVAC-HIV, which is a recombinant canarypox vaccine express HIV-1 Gag, Pro and gp120-gp41, plus two boosting intramuscular injections of AIDSVAX® B/E, which is an alum-adjuvanted bivalent HIV-1 gp120 vaccine of subtypes E and B [24]. Though the low prevention rate of vaccination with ALVAC/AIDSVAX combination excludes the products from approval for clinical prophylaxis of HIV, the moderate effects displayed in human trial not only provoked scientists to make deep explorations on the causes for the failures, but also presented researchers a great encouragement to commit further efforts to developing efficacious HIV vaccines. Subsequently, based on the virion features which are more and more clearly elaborated in structure and function, scientists set out to handle the obstacles identified to the development of a preventive HIV vaccine from several aspects, such as accurately targeting the conserved antigenic proteins, seeking Ags able to induce the broadly neutralizing Abs (bnAbs), and formulating a highly efficient VADS [25]. As mentioned above, a VLP-based VADS proves a highly potent inducer for Abs and helper T cell responses and also able to elicit robust cytotoxic T cell responses necessary for preventing primary infections and erasing infected cells, thus offering researchers an alternative tool to engineer effective vaccines against HIV, which is regarded as the most challenging foe owing to its poor immunogenicity, fragile surface glycoprotein, and the ability to overpower the cell immune system [26].

HEK293 cell (Human embryonic kidney 293 cell) expression system, Salvo et al*.* engineered a VLP-based VADS composed of Zika prM/E (pre-membrane and envelope) glycoproteins for making vaccines to defend against Zika and demonstrated that mice injected with Zika VLP combined with adjuvant alum secreted high levels of the Ag-neutralizing Abs [31]. In particular, the vaccinated mice all survived without morbidity or weight loss after receiving the lethal challenge with the dose of 200 PFU of Zika strain H/PF/2013, proving the protective efficacy of the VLP-based Zika vaccine which may be tested in humans as a prophylactic candidate with minimal safety concerns to protect unborn babies whose mothers become

Polymeric Nanoparticles Engineered as a Vaccine Adjuvant-Delivery System

http://dx.doi.org/10.5772/intechopen.81084

33

Similarly, Espinosa and colleagues formulated a ZIKV vaccine based on virus-like particles (VLPs) which were generated in HEK293 cells transiently transfected with the prM/E genes of Zika placed downstream from a heterologous signal sequence and observed efficient induction of neutralizing antibody and a dose-sparing effect of alum in VLP-immunized mice (C57Bl/6 x Balb/c) [32]. In addition, passive transfer experiments showed that AG129 mice received the sera from immunized mice prior to Zika infection manifested significantly reduced viral replication as indicated by viral RNA levels in the blood and successfully conquered the infection to contrast control mice which succumbed to infection, underscoring the protective effect of the humoral immunity elicited by this VLP-based Zika vaccine candidate. In summary, the VLP-based VADSs are a potent inducer of Ab and cellular responses and also possesses the prerequisite features required to prepare the vaccines that are able not only to prevent the primary infections but also to clear infected cells, thus representing an alternative tool promising to engineer efficacious vaccines against the intractable pathogens, such as

Chitosan, a linear polysaccharide composed of randomly distributed β-(1,4)-linked D-glucosamine (deacetylated unit) and N-acetyl-D-glucosamine (acetylated unit), is usually made through hydrolysis of the chitin shells of shrimp and other crustaceans with an alkaline substance, such as sodium hydroxide [34]. Chitosan has a positive charge under neutral conditions due to protonation of basic amine groups, which contributes to the enhancement of solubility of the compound; however, the dissolution behavior of chitosan in aqueous media is also negatively influenced by the number and structural form of acetylated groups, allowing chitosan able to be used for agent delivery in several distinct forms, including solution, hydrogel, and especially nano/microparticle, which can be obtained via crosslinking, ionotropic gelation or precipitation-coacervation [35]. Interestingly, novel chitosan derivatives with customized biochemical properties are still continuously synthesized through facile conjugation of side chain moieties of functional molecules to solvent-accessible amine and hydroxyl groups, rapidly expanding chitosan in application range and dosage form [36].

In retrospect, in 1980s, researchers observed that chitosan of 70% deacetylated chitin could activate peritoneal macrophages [37] and induce production of various cytokines in mice [38], which was subsequently further explored by Illum et al*.* to demonstrate that in mouse model chitosan, via nasal immunization, could act as an efficient VADS able to remarkably enhance

infected with Zika during pregnancy.

HCV, HIV, and even parasites [33].

**2.3. Chitosan NPs**

Recently, Chapman et al*.* constructed an MVA (modified vaccinia Ankara)-mGag (an HIV-1 subtype C mosaic Gag immunogen) and a DNA-mGag vaccine, which were designed to address the tremendous diversity of HIV, and showed that mGag budded from cells infected and transfected with MVA-mGag and DNA-mGag, respectively, formed VLPs [27]. In mice, the DNA-mGag homologous prime boost vaccination elicited predominantly CD8+ T cells, and the homologous MVA-mGag vaccination induced predominantly CD4+ T cells; in contrast, a heterologous DNA-mGag prime MVA-mGag boost induced strong, more balanced Gag CD8+ and CD4+ T cell responses that were predominantly of an effector memory phenotype. Also, it was found that DNA-mGag homologous vaccination induced much higher cumulative Ag-specific IFN-γ secretion responses and generated significant higher levels of cytokine-positive CD8+ T cells than DNA-nGag (natural Gag), indicating a heterologous prime-boost regimen with DNA and MVA vaccines expressing HIV-1 subtype C mosaic Gag as an Ag is highly immunogenic and may be an effective VLP-based VADS for eliciting strong immunity to HIV. Yao's group formulated a VLP-based HIV vaccine, which was composed of HIVIIIB Gag and HIVBaL gp120/ gp41 envelope as a pseudovirion vaccine capable of presenting Ags in their native conformations and was engineered through using HEK (human embryonic kidney cell)-derived cell line expression system [28]. The researchers demonstrated that mice vaccinated by intranasal prime followed by two sub-cheek boosts with VLPs adjuvanted with liposomes entrapping TLR3 ligand dsRNA were stimulated to secrete high titers of Abs against the Ags, with predominant IgG2c over IgG and produce a significantly increased germinal center B cells and T follicular cells, suggesting that the VLP-based VADS is superior for induction of a Th1-biased immune response, while prolonging lymph node germinal centers, T follicular cells, and generating neutralizing antibodies, and thus is rather suitable for making HIV vaccines [26].

Notably, certain types of pathogens that are once known to cause only a mild and self-healing illness and therefore never listed in dangerous items and may abruptly cause the unexpected problems associated with human and population health, hinting the existence of undisclosed infection mechanisms and pathophysiological processes or the emergence of mutations relevant to severe toxicity. For example, during the 2015–2016 South American Zika epidemic, the mosquito-borne virus which used to cause mild symptoms, such as fever, skin rash, and joint or muscle pain, was eventually identified able to cause severe damage to fetal brain through infecting pregnant women and thus finally recognized as the culprit responsible for thousands of microcephaly affected new borns, raising a great social problem and concern [29]. Unfortunately, up to now, still there are no licensed vaccines for prophylaxis of Zika, though several conventional approaches have been tried on developing such as an urgently needed products, including inactivated, recombinant live-attenuated viruses, protein subunit vaccines, RNA and DNA vaccines, as well as the VLP-based VADS [30]. Recently, using HEK293 cell (Human embryonic kidney 293 cell) expression system, Salvo et al*.* engineered a VLP-based VADS composed of Zika prM/E (pre-membrane and envelope) glycoproteins for making vaccines to defend against Zika and demonstrated that mice injected with Zika VLP combined with adjuvant alum secreted high levels of the Ag-neutralizing Abs [31]. In particular, the vaccinated mice all survived without morbidity or weight loss after receiving the lethal challenge with the dose of 200 PFU of Zika strain H/PF/2013, proving the protective efficacy of the VLP-based Zika vaccine which may be tested in humans as a prophylactic candidate with minimal safety concerns to protect unborn babies whose mothers become infected with Zika during pregnancy.

Similarly, Espinosa and colleagues formulated a ZIKV vaccine based on virus-like particles (VLPs) which were generated in HEK293 cells transiently transfected with the prM/E genes of Zika placed downstream from a heterologous signal sequence and observed efficient induction of neutralizing antibody and a dose-sparing effect of alum in VLP-immunized mice (C57Bl/6 x Balb/c) [32]. In addition, passive transfer experiments showed that AG129 mice received the sera from immunized mice prior to Zika infection manifested significantly reduced viral replication as indicated by viral RNA levels in the blood and successfully conquered the infection to contrast control mice which succumbed to infection, underscoring the protective effect of the humoral immunity elicited by this VLP-based Zika vaccine candidate.

In summary, the VLP-based VADSs are a potent inducer of Ab and cellular responses and also possesses the prerequisite features required to prepare the vaccines that are able not only to prevent the primary infections but also to clear infected cells, thus representing an alternative tool promising to engineer efficacious vaccines against the intractable pathogens, such as HCV, HIV, and even parasites [33].
